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6 followed by a frozen embryo transfer (FET) results in high implantation rate (IR), clinical pregnancy rate (CPR) and is superior to day 3 biopsy
P-78 Tuesday, October 23, 2012 TROPHECTODERM (TE) BIOPSY WITH VITRIFICATION ON DAY 5/6 FOLLOWED BY A FROZEN EMBRYO TRANSFER (FET) RESULTS IN HIGH IMPLANTATION RATE (IR), CLINICAL PREGNANCY RATE (CPR) AND IS SUPERIOR TO DAY 3 BIOPSY. H.-L. Lee, A. Adler, E. Ampeloquio, T.-L. Lee, A. Berkeley, J. Grifo. NYUFC, New York Langone Medical Center, New York, NY. OBJECTIVE: To compare the clinical outcomes for TE biopsy vs. day 3 embryo biopsy (EB) for preimplantation genetic diagnosis (PGD) of single gene mutations or preimplantation genetic screening (PGS) of all 24 chromosomes including translocations. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: A total of 185 patients underwent an IVF cycle with PGD or PGS. Group A (n¼96; mean maternal age 36.5) underwent EB on day 3 and only euploid or embryos not affected with genetic disease had embryo transfer (ET) on day 5. Group B (n¼89; mean maternal age 35) underwent TE biopsy on days 5 and/or 6. Biopsied blastocysts were vitrified shortly after biopsy and only euploid or unaffected blastocysts were transferred in a subsequent FET. RESULTS: In Group A, 164 embryos were found to be euploid or unaffected by genetic disease and suitable for fresh ET on day 5 (mean¼1.7 embryos/ET). In Group B, 153 vitrified embryos were warmed and transferred (mean¼1.4 embryos per FET). There was a significantly higher IR of 48% (74/153) following TE biopsy and vitrification as compared to day 3 EB 36% (59/164) (P¼0.0232). Also a significantly higher CPR expressed per retrieval cycle was achieved following warming and FET of TE biopsy with 65% (58/89) pregnancy rate with fetal hearts as compared to 43% (41/96) for the day 3 EB (P¼0.0031). CONCLUSION: PGD/PGS analysis of multiple cells biopsied from the TE of blastocysts on days 5 or 6 displayed significantly higher IR and CPR when compared to day 3 EB. Despite vitrification and warming in subsequent cycle, the euploid/unaffected blastocysts transferred following TE biopsy appear to be more viable than those that have day 3 EB with fresh ET. These results validate TE biopsy being a more efficient tool to identify euploid embryos and achieve viable pregnancies.
P-79 Tuesday, October 23, 2012 SUCCESSFUL APPLICATION OF SIMULTANEOUS CHROMOSOMAL MICROARRAY ANALYSIS (CMA) AND GENETIC DISEASE DIAGNOSIS FROM DAY 5 TROPHECTODERM (TE) EMBRYO BIOPSIES WITH DAY 6 EMBRYO TRANSFER. K. D. Tran, B. D. Mariani, S. L. Shrestha, M. C. Sands, V. Novik, H. J. Stern. Genetics and IVF Institute, Fairfax, VA. OBJECTIVE: To use CMA combined with mutation-specific PCR protocols designed for various mutational classes for the detection of unaffected euploid embryos from couples at risk for genetic disease. DESIGN: Prospective proof of concept. MATERIALS AND METHODS: Whole genome amplification of TE biopsies was performed using the SurePlex DNA Amplification System and analyzed by 24sure v3 arrays (BlueGnome). Aliquots of SurePlex amplified DNA products were subjected to laboratory-developed fluorescent PCR coupled with capillary electrophoresis for genetic disease diagnosis. Mutational detection of single base substitutions and small indels were performed using the ABI SNaPshotÒ kit with laboratory-developed primers. Larger genetic lesions were detected by PCR fragment length analysis. STR markers linked to the mutation of interest were incorporated into all assays. RESULTS: No modification of the CMA protocol was necessary and no pre-amplification steps prior to SurePlex DNA amplification were required. SurePlex amplified DNA products were subjected to single round PCR for mutational and linked marker detection, except for mutations involving triplet repeats where nested PCR was necessary. Marker alleles and patient-specific mutational PCR profiles were consistent between blood DNA controls and related TE DNA products in all cases. Our group has successfully validated this methodology and performed several clinical cases for a variety of genetic lesions, including substitutions, indels, and triplet repeats. CONCLUSION: Our study supports the proof of concept for combining CMA with mutation-specific PCR for testing Day 5 TE biopsies in a timeframe allowing for a Day 6 fresh transfer. The combination of these important techniques provides a comprehensive approach that now can be offered to couples at risk for single-gene disorders. Our combined protocol can detect embryos unaffected with single-gene disorders caused by a variety of muta-
FERTILITY & STERILITYÒ
tional classes and simultaneously determine if those embryos are free of chromosomal aneuploidy.
P-80 Tuesday, October 23, 2012 DELIVERY OF A CHROMOSOMALLY NORMAL BABY AFTER THE TRANSFER OF AN EMBRYO DIAGNOSED AS ANEUPLOID BY 24-CHROMOSOME PREIMPLANTATION GENETIC SCREENING. C. A. Guerrero, A. Fleming, J. S. Goldstein. Fertility Specialists of Texas, Frisco, TX. OBJECTIVE: To demonstrate that a day 3 embryo diagnosed as aneuploid by preimplantation genetic screening is capable of producing a chromosomally normal child. DESIGN: A case report. MATERIALS AND METHODS: A 34 year old Gravida 1, Para 1 delivered a son diagnosed with Persistant Mullerian Duct Syndrome (PMDS). Patient elected for Preimplantation Genetic Screening (PGS) for female karyotype and underwent In Vitro Fertilization. PGS of day 3 embryos was performed using single nucleotide polymorphism (SNP) microarray based 24-chromosome screening. A total of 9 embryos were biopsied and blastomeres sent off for analysis. RESULTS: One of the nine embryos was diagnosed as a normal 46;XX. The remaining 8 embryos were reported as aneuploid. The euploid embryo had poor morphological quality on day 3 and did not develop to the blastocyst stage. Patient was counseled not to transfer any of the remaining abnormal embryos, however, elected to transfer the highest quality blastocyst from the cohort of embryos regardless of the chromosome screening report. One good quality blastocyst reported as an aneuploid 42; XX; Nullisomy 8; Monosomy 7, 18 was transferred. A clinical pregnancy was reported with normal fetal heart motion. Chorionic villus sampling analysis revealed no evidence of chromosome abnormalities in the fetus confirming a normal 46; XX karyotype. A healthy baby was born at 36 weeks weighing 6.1 lbs and 18 3/4 inches long. CONCLUSION: This outcome indicates that normal embryos are possibly being misdiagnosed as abnormal by the commonly used preimplantation genetic screening of day 3 embryos. This, in turn, raises concern about the false positive rate being more elevated than what traditionally has been reported. Furthermore, it further solidifies the importance of moving on to day 5 trophectoderm biopsy to prevent the discarding of normal embryos. Aneuploidy screening of day 3 embryos should not be routinely offered to patients and should be used with caution.
P-81 Tuesday, October 23, 2012 INCIDENCE OF ANEUPLOIDY IS INCREASED IN PATIENTS WITH ADVANCED MATERNAL AGE AND RECURRENT PREGNANCY LOSS. L. Guzman,a,b B. Acacio,c B. S. Shapiro,d M. Perloe,e W. Venier,f T. Escudero.g aReprogenetics Latinoamerica, Lima, Peru; b Grupo Pranor, Lima, Peru; cAcacio Fertility, California; dFertility Center of Las Vegas, Las Vegas, NV; eGeorgia Reproductive Specialists, Atlanta, GA; fSan Diego Fertility Center, San Diego, CA; gReprogentics, Livingston, NJ. OBJECTIVE: To determine the frequency of aneuploidy in preimplantational embryos derived from advanced maternal age (AMA), recurrent pregnancy loss (RPL), spontaneous abortion (SAB) and patient request (RQ) according to the day of embryo biopsy. DESIGN: Multicenter observational study. MATERIALS AND METHODS: Embryo biopsies on day 3 or day 5 were referred from fertility centers to the same PGD laboratory. The frequency of aneuploidy in embryos was analyzed according to the patient reason to perform array CGH test. AMA patients were R38 years old. Only patients%35 years old (young women) were selected for RPL, SAB and RQ patients to avoid the influence of the age on the frequency of aneuploidy. RESULTS: A total of 1703 IVF/ICSI cycles were selected. Embryo biopsies were done at day 3 (1109 cycles) or day 5 (594 cycles). Average age was higher in AMA patient (40.72.1); RPL, SAB and RQ age were similar (32.22.8, 31.92.7 and 31.71.7 years old respectively). On day 3, AMA patient had 18.2% (911/5015) of euploid embryos compared to 43.7% (1158/2649) in young women (P<0.001). Euploidy rates on day 3 were lower for RPL 41.5% (350/841) and SAB 41.5% (248/597) groups compared to RQ patients 46.2% (560/1211) (P<0.05). On day 5,
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P-79 Tuesday, October 23, 2012 SUCCESSFUL APPLICATION OF SIMULTANEOUS CHROMOSOMAL MICROARRAY ANALYSIS (CMA) AND GENETIC DISEASE DIAGNOSIS FROM DAY 5 TROPHECTODERM (TE) EMBRYO BIOPSIES WITH DAY 6 EMBRYO TRANSFER. K. D. Tran, B. D. Mariani, S. L. Shrestha, M. C. Sands, V. Novik, H. J. Stern. Genetics and IVF Institute, Fairfax, VA. OBJECTIVE: To use CMA combined with mutation-specific PCR protocols designed for various mutational classes for the detection of unaffected euploid embryos from couples at risk for genetic disease. DESIGN: Prospective proof of concept. MATERIALS AND METHODS: Whole genome amplification of TE biopsies was performed using the SurePlex DNA Amplification System and analyzed by 24sure v3 arrays (BlueGnome). Aliquots of SurePlex amplified DNA products were subjected to laboratory-developed fluorescent PCR coupled with capillary electrophoresis for genetic disease diagnosis. Mutational detection of single base substitutions and small indels were performed using the ABI SNaPshotÒ kit with laboratory-developed primers. Larger genetic lesions were detected by PCR fragment length analysis. STR markers linked to the mutation of interest were incorporated into all assays. RESULTS: No modification of the CMA protocol was necessary and no pre-amplification steps prior to SurePlex DNA amplification were required. SurePlex amplified DNA products were subjected to single round PCR for mutational and linked marker detection, except for mutations involving triplet repeats where nested PCR was necessary. Marker alleles and patient-specific mutational PCR profiles were consistent between blood DNA controls and related TE DNA products in all cases. Our group has successfully validated this methodology and performed several clinical cases for a variety of genetic lesions, including substitutions, indels, and triplet repeats. CONCLUSION: Our study supports the proof of concept for combining CMA with mutation-specific PCR for testing Day 5 TE biopsies in a timeframe allowing for a Day 6 fresh transfer. The combination of these important techniques provides a comprehensive approach that now can be offered to couples at risk for single-gene disorders. Our combined protocol can detect embryos unaffected with single-gene disorders caused by a variety of muta-
FERTILITY & STERILITYÒ
tional classes and simultaneously determine if those embryos are free of chromosomal aneuploidy.
P-80 Tuesday, October 23, 2012 DELIVERY OF A CHROMOSOMALLY NORMAL BABY AFTER THE TRANSFER OF AN EMBRYO DIAGNOSED AS ANEUPLOID BY 24-CHROMOSOME PREIMPLANTATION GENETIC SCREENING. C. A. Guerrero, A. Fleming, J. S. Goldstein. Fertility Specialists of Texas, Frisco, TX. OBJECTIVE: To demonstrate that a day 3 embryo diagnosed as aneuploid by preimplantation genetic screening is capable of producing a chromosomally normal child. DESIGN: A case report. MATERIALS AND METHODS: A 34 year old Gravida 1, Para 1 delivered a son diagnosed with Persistant Mullerian Duct Syndrome (PMDS). Patient elected for Preimplantation Genetic Screening (PGS) for female karyotype and underwent In Vitro Fertilization. PGS of day 3 embryos was performed using single nucleotide polymorphism (SNP) microarray based 24-chromosome screening. A total of 9 embryos were biopsied and blastomeres sent off for analysis. RESULTS: One of the nine embryos was diagnosed as a normal 46;XX. The remaining 8 embryos were reported as aneuploid. The euploid embryo had poor morphological quality on day 3 and did not develop to the blastocyst stage. Patient was counseled not to transfer any of the remaining abnormal embryos, however, elected to transfer the highest quality blastocyst from the cohort of embryos regardless of the chromosome screening report. One good quality blastocyst reported as an aneuploid 42; XX; Nullisomy 8; Monosomy 7, 18 was transferred. A clinical pregnancy was reported with normal fetal heart motion. Chorionic villus sampling analysis revealed no evidence of chromosome abnormalities in the fetus confirming a normal 46; XX karyotype. A healthy baby was born at 36 weeks weighing 6.1 lbs and 18 3/4 inches long. CONCLUSION: This outcome indicates that normal embryos are possibly being misdiagnosed as abnormal by the commonly used preimplantation genetic screening of day 3 embryos. This, in turn, raises concern about the false positive rate being more elevated than what traditionally has been reported. Furthermore, it further solidifies the importance of moving on to day 5 trophectoderm biopsy to prevent the discarding of normal embryos. Aneuploidy screening of day 3 embryos should not be routinely offered to patients and should be used with caution.
P-81 Tuesday, October 23, 2012 INCIDENCE OF ANEUPLOIDY IS INCREASED IN PATIENTS WITH ADVANCED MATERNAL AGE AND RECURRENT PREGNANCY LOSS. L. Guzman,a,b B. Acacio,c B. S. Shapiro,d M. Perloe,e W. Venier,f T. Escudero.g aReprogenetics Latinoamerica, Lima, Peru; b Grupo Pranor, Lima, Peru; cAcacio Fertility, California; dFertility Center of Las Vegas, Las Vegas, NV; eGeorgia Reproductive Specialists, Atlanta, GA; fSan Diego Fertility Center, San Diego, CA; gReprogentics, Livingston, NJ. OBJECTIVE: To determine the frequency of aneuploidy in preimplantational embryos derived from advanced maternal age (AMA), recurrent pregnancy loss (RPL), spontaneous abortion (SAB) and patient request (RQ) according to the day of embryo biopsy. DESIGN: Multicenter observational study. MATERIALS AND METHODS: Embryo biopsies on day 3 or day 5 were referred from fertility centers to the same PGD laboratory. The frequency of aneuploidy in embryos was analyzed according to the patient reason to perform array CGH test. AMA patients were R38 years old. Only patients%35 years old (young women) were selected for RPL, SAB and RQ patients to avoid the influence of the age on the frequency of aneuploidy. RESULTS: A total of 1703 IVF/ICSI cycles were selected. Embryo biopsies were done at day 3 (1109 cycles) or day 5 (594 cycles). Average age was higher in AMA patient (40.72.1); RPL, SAB and RQ age were similar (32.22.8, 31.92.7 and 31.71.7 years old respectively). On day 3, AMA patient had 18.2% (911/5015) of euploid embryos compared to 43.7% (1158/2649) in young women (P<0.001). Euploidy rates on day 3 were lower for RPL 41.5% (350/841) and SAB 41.5% (248/597) groups compared to RQ patients 46.2% (560/1211) (P<0.05). On day 5,
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